TNFalpha is a primary cytokine responsible for inflammatory and immunosuppressive responses in skin. After UV-B irradiation of cultured human keratinocytes, we found that TNFalpha was released into the media, as monitored by ELISA, and was bound to cells, as observed by immunofluorescence microscopy. The release of TNFalpha into cell culture supernatant during the 24 h after UV-B irradiation was augmented by the addition of IL-1alpha to the cells. Further, we found this secretion was unaffected by rapamycin, and therefore independent of FRAP DNA-protein kinase mediated signal transduction. However, UV-B also induced expression of membrane-bound TNFalpha, and this was dependent on FRAP signaling. In wild type mice, TNFalpha bound to skin increased immediately after irradiation, declined at 6 h, and then rose again at 12 h before falling by 24 h. This pattern of induction was confirmed by RT-PCR of TNFalpha mRNA message in cultured epidermal cells. Induction of membrane-bound TNFalpha was also found in c-fos gene knockout mice deficient in the AP-1 transcription factor, suggesting that, although AP-1 containing c-fos signaling is required for some UV responses, AP-1 containing c-fos is not required for this TNFalpha activation. However, in homozygous p53 knockout mice the basal level of TNFalpha bound to the epidermis was greatly elevated without UV irradiation. This level declined and remained constant following irradiation. This implies that p53 directly or indirectly represses TNFalpha gene expression and that modification of p53 mRNA stability or phosphorylation of p53 protein after UV may be responsible for TNFalpha induction in the membrane. Overexpression of the immunosuppressive cytokine TNFalpha in this locale may contribute to the carcinogen-susceptibility of p53 knockout mice.
O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl adducts from DNA and may be important in tumor resistance to alkylation chemotherapy. MGMT was visualized in human cells and tumor tissues with monoclonal antibodies against MGMT and immunofluorescence microscopy, and fluorescent signals were quantified by digital image analysis. MGMT was found both in the cytoplasm and the nucleus, and in either locale the protein reacts with alkylated DNA bases and becomes inactivated and lost from the cell. Cell lines in culture and xenografts showed a broad normal distribution of nuclear MGMT levels, but human brain tumors often showed a skewed distribution, with a significant fraction of cells with high levels of MGMT. O(6)-Benzylguanine, a suicide substrate inactivator for MGMT activity, reduced MGMT in human cells and in a mouse xenograft to levels undetectable by antibody assay 1 h post-treatment. In melanoma specimens taken from a patient 3 h post-treatment with temozolomide, MGMT levels were reduced by 70%. This quantitative immunofluorescence assay can be used to monitor MGMT and it depletion in human tumors to improve the use of alkylating agents in cancer chemotherapy.
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